Rapid Detection of Plant Bacterial Wilt by Loop-mediated Isothermal Amplification

doi:10.13560/j.cnki.biotech.bull.1985.2020-0389

Abstract

Abstract:

The purpose of the study is to establish a rapid detection method for Ralstonia solanacearum,which is beneficial to the early diagnosis of plant bacterial wilt in the field. The LAMP（loop-mediated isothermal amplification）method for amplification of cytochrome C gene was established by optimization of reaction conditions. The minimum detection limit with the LAMP method reached 1 pg. The detection could be completed within 1 h without using complicated instruments and the results were visible by human eye. Using this method,R. solanacearum was detected in the stem extracts of manually inoculated eggplant,tomato,peanut,sesame and Amaranthus lividus and potato tuber tissue fluid of potato,especially,it was appropriate for the detection of sesame,peanut,tomato,potato and sweet potato plants suspected of being in disease in the field,and the detection rate by LAMP was much higher than that by PCR method. The LAMP method is a fast,specific,sensitive,and easy-to-use for the field detection of R. solanacearum bacterial wilt,which is suitable for promotion at the grassroots level.

Key words: Ralstonia solanacearum, loop-mediated isothermal amplification, rapid detection

LI Xin-shen, HUANG Xiao-mei, WU Shu-xiu, HUANG Rui-rong, WEI Lin-gen, HUA Ju-ling. Rapid Detection of Plant Bacterial Wilt by Loop-mediated Isothermal Amplification[J]. Biotechnology Bulletin, 2021, 37(1): 272-281.

Figures/Tables 8

References 51

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菌株编号	菌株名称	寄主	演化型/生理小种
1	青枯病菌菌株Ejxnc01	茄子	Ⅰ/1
2	青枯病菌菌株Cjxnc01	辣椒	Ⅰ/1
3	青枯病菌菌株Tjxjx01	番茄	Ⅰ/1
4	青枯病菌菌株Pjxga01	花生	Ⅰ/1
5	青枯病菌菌株Seppx05	芝麻	Ⅰ/1
6	青枯病菌菌株JSja03	凹头苋	Ⅰ/1
7	青枯病菌菌株Tjxga03	烟草	Ⅰ/1
8	青枯病菌菌株Gjxjj01	姜	Ⅰ/4
9	青枯病菌菌株Pjxnc01	马铃薯	Ⅱ/3
10	青枯病菌菌株Pjxga02	马铃薯	Ⅱ/3
11	青枯病菌菌株Pjxjj03	马铃薯	Ⅱ/3
12	青枯病菌菌株Pjxda04	马铃薯	Ⅱ/3
13	蒲桃雷尔氏菌R001	丁香树	-
14	皮氏伯克霍尔德氏菌Bp001	土壤	-

菌株编号	菌株名称	寄主	演化型/生理小种
1	青枯病菌菌株Ejxnc01	茄子	Ⅰ/1
2	青枯病菌菌株Cjxnc01	辣椒	Ⅰ/1
3	青枯病菌菌株Tjxjx01	番茄	Ⅰ/1
4	青枯病菌菌株Pjxga01	花生	Ⅰ/1
5	青枯病菌菌株Seppx05	芝麻	Ⅰ/1
6	青枯病菌菌株JSja03	凹头苋	Ⅰ/1
7	青枯病菌菌株Tjxga03	烟草	Ⅰ/1
8	青枯病菌菌株Gjxjj01	姜	Ⅰ/4
9	青枯病菌菌株Pjxnc01	马铃薯	Ⅱ/3
10	青枯病菌菌株Pjxga02	马铃薯	Ⅱ/3
11	青枯病菌菌株Pjxjj03	马铃薯	Ⅱ/3
12	青枯病菌菌株Pjxda04	马铃薯	Ⅱ/3
13	蒲桃雷尔氏菌R001	丁香树	-
14	皮氏伯克霍尔德氏菌Bp001	土壤	-

引物名称	引物序列（5'-3'）	长度/bp
F3	AGCGGTGCCAATCCGTA	17
B3	TGCCATGGTCAGGTACTGAT	20
FIP	AGCAATCCGAAGGTGCCGAATGTCGCGTA- CAACCAGGA	38
BIP	TCGGTATCCCGACAACACCATGTGGGCGT- CGATCGCATA	39

引物名称	引物序列（5'-3'）	长度/bp
F3	AGCGGTGCCAATCCGTA	17
B3	TGCCATGGTCAGGTACTGAT	20
FIP	AGCAATCCGAAGGTGCCGAATGTCGCGTA- CAACCAGGA	38
BIP	TCGGTATCCCGACAACACCATGTGGGCGT- CGATCGCATA	39

来源植物	取样部位	样本数量	LAMP检测		PCR检测
来源植物	取样部位	样本数量	检出总数	检出率	检出总数	检出率
芝麻	茎杆	25	25	100%	23	92.00%
花生	茎杆	24	23	95.83%	21	87.50%
番茄	叶片	23	23	100%	21	91.30%
马铃薯	块茎	24	23	95.83%	22	91.67%
甘薯	块茎	22	21	95.45%	20	90.91%